EP4232454A1 - Compositions et procédés de traitement d'états ischémiques - Google Patents

Compositions et procédés de traitement d'états ischémiques

Info

Publication number
EP4232454A1
EP4232454A1 EP21883794.6A EP21883794A EP4232454A1 EP 4232454 A1 EP4232454 A1 EP 4232454A1 EP 21883794 A EP21883794 A EP 21883794A EP 4232454 A1 EP4232454 A1 EP 4232454A1
Authority
EP
European Patent Office
Prior art keywords
mann
cells
subject
cell
vegf
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21883794.6A
Other languages
German (de)
English (en)
Other versions
EP4232454A4 (fr
Inventor
Napoleone Ferrara
Cuiling Zhong
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of California
University of California Berkeley
University of California San Diego UCSD
Original Assignee
University of California
University of California Berkeley
University of California San Diego UCSD
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of California, University of California Berkeley, University of California San Diego UCSD filed Critical University of California
Publication of EP4232454A1 publication Critical patent/EP4232454A1/fr
Publication of EP4232454A4 publication Critical patent/EP4232454A4/fr
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7008—Compounds having an amino group directly attached to a carbon atom of the saccharide radical, e.g. D-galactosamine, ranimustine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • the present disclosure generally relates to compositions and methods for treating ischemic conditions.
  • Angiogenesis is a complex process involving the growth of new blood vessels from the existing vasculature and occurs in both physiological and pathological circumstances. In tumors, angiogenesis facilitates rapid growth and metastasis through delivery of nutrients and oxygen and removal of metabolic wastes [1], Development of the vasculature requires the coordinated activation of multiple signaling pathways, including VEGF/VEGFR, angiopoietin (Ang)/Tie2, Notch, Ephrin/Eph and PDGF/PDGFR [2, 3], Stimulating angiogenesis has the potential of facilitating treatment of a number of conditions characterized by reduced perfusion, including diabetic ulcers, myocardial and limb ischemia [4, 5], Conversely, blocking angiogenesis is a clinically validated strategy to treat malignant tumors and intraocular neovascular disorders [1, 6].
  • Endothelial cell (EC) metabolism is hypothesized to play a key role in the regulation of angiogenesis in normal and pathological circumstances.
  • Metabolic switches in ECs such as fatty acid, glucose, and glutamine metabolism, have been reported to trigger angiogenesis [7, 8]
  • ECs in the tumor vasculature are known to rely on glycolysis for ATP production, for instance through enhanced expression of glucose transporter GLUTE Lowering glycolysis in tumor ECs arrests their proliferation [9]
  • aberrant glycosylation patterns have been documented during oncogenic transformation and progression of cancer and it has been proposed that inhibiting glycosylation may result in suppression of key angiogenesis pathways, including VEGF/VEGFR2 and Notch [10]
  • Evidence that has emerged in recent years points to glycans as novel angiogenesis regulators due to changes in protein glycosylation [11],
  • the gly can-binding protein Galectinl has been reported to interact with VEGFR2, leading to ligand-independent receptor activation
  • compositions and methods for treating an ischemic condition in a subject includes hexosamine D-mannosamine (ManN).
  • a method for treating an ischemic condition in a subject includes administering to the subject in need thereof an effective amount of hexosamine D-mannosamine (ManN).
  • the administration is effective to promote endothelial cell proliferation and angiogenesis in the subject.
  • the method further includes administering to the subj ect in need thereof an effective amount of an N-gly cosylation inhibitor.
  • the method further includes administering to the subject in need thereof an effective amount of VEGF.
  • the ischemic condition is caused by a disease or a trauma.
  • the administration is intravenous, intraperitoneal, or intravitreal.
  • the present disclosure provides pharmaceutical compositions and methods for inducing angiogenesis in a subject, including administering to the subject in need thereof an effective amount of hexosamine D-mannosamine (ManN).
  • ManN hexosamine D-mannosamine
  • the administration is effective to reduce ischemia in the subject.
  • the method further includes administering to the subject in need thereof an effective amount of an N-glycosylation inhibitor.
  • the method further includes administering to the subject in need thereof an effective amount of VEGF.
  • the subject is in need of inducing angiogenesis due to an ischemic condition is caused by a disease or a trauma.
  • the administration is intravenous, intraperitoneal, or intravitreal.
  • the present disclosure provides pharmaceutical compositions and methods for inhibiting protein glycosylation in a cell, including administering to the cell an effective amount of hexosamine D-mannosamine (ManN).
  • ManN hexosamine D-mannosamine
  • the administration is in vivo. In embodiments, the administration is ex vivo. In embodiments, the administration is effective to stimulate EC proliferation and angiogenesis. In embodiments, the administration is effective to activate JNK and an unfolded protein response caused by ER stress. In embodiments, the administration is effective to induce changes in N-glycan and O-glycan profiles.
  • FIGS. 1A to IF represent examples showing the effects of Mannosamine (ManN) on bovine choroidal microvascular endothelial cells (BCEC) proliferation.
  • FIG. 1 A is an image showing crystal-violet stained BCEC samples treated with ManN, in the presence or absence of VEGF. BCECs were treated with various concentrations of ManN ranging from 0.5 pM to 1 mM for 5-6 days, with or without 5ng/ml VEGF. At the end of the experiment, cells were fixed and stained with crystal violet. Cell-covered areas in various treatment groups were quantified by ImageJ software.
  • FIG. IB is a chart showing the effect of ManN in the presence or absence of VEGF on cell numbers.
  • FIG. ID is an image showing effects of hexosamines other than ManN on BCEC proliferation in samples. Each treatment group was tested in duplicate.
  • FIGS. 2 A to 2C are western blot images showing activation of ERK, AKT, mTOR, AMPKa, CREB, ACC, and eNOS is not unique to ManN.
  • BCECs were treated with 40 pM ManN, ManNAc or mannose for various times.
  • FIGS. 3 A to 3E represent examples showing that ManN specifically activates the JNK pathway in BCECs.
  • FIG. 3 A is a western blot image. BCECs grown in Growth Media (GM: low glucose DMEM containing 10% bovine calf serum (BCS), 10 ng/ml VEGF and 5 ng/ml bFGF) were switched to growth factor-free media, followed by treatment with ManN or Mannose at 4 pM-4 mM.
  • GM low glucose DMEM containing 10% bovine calf serum (BCS), 10 ng/ml VEGF and 5 ng/ml bFGF
  • FIG. 3B is a western blot image showing that ManN, but not mannose, could activate JNK and its downstream c-Jun. P-actin served as the loading control. For each study, a representative experiment is shown from 2-3 independent studies.
  • FIG. 3C is a chart showing the effect of ManN on pre-treated samples.
  • FIG. 3D is a western blot image showing screening of siRNAs against JNK1 and JNK2. 24 hours after siRNA transfection, BCECs were lysed and proteins were subjected to western blot analysis. P-actin served as the loading control. Quantification of target knockdown is shown.
  • FIGS. 4A to 4G represent examples showing that ManN affects protein glycosylation.
  • FIG. 4A is a western blot image showing reduction of VEGFR2 molecular mass following ManN treatment. BCECs were treated with various hexosamines, their derivatives and monosaccharides at 40 pM or with VEGF at 5 ng/ml for 24 hours. VEGFR2 western blot analysis was performed.
  • FIG. 4B is a western blot imaging showing dose-dependent effects of ManN on VEGFR2 molecular mass in BCECs.
  • FIG. 4A is a western blot image showing reduction of VEGFR2 molecular mass following ManN treatment. BCECs were treated with various hexosamines, their derivatives and monosaccharides at 40 pM or with VEGF at 5 ng/ml for 24 hours. VEGFR2 western blot analysis was performed.
  • FIG. 4B is a western blot imaging showing dose-dependent effects of ManN
  • FIG. 4C is a western blot image showing Mannose could dose-dependently reverse the effect of 2 mM ManN on VEGFR2 molecular mass change, whereas mannose alone had no effect even at 10 mM.
  • FIG. 4D is a chart showing that 5 mM mannose could completely reverse the bell-shaped effects of ManN on BCEC proliferation with or without 5 ng/ml VEGF.
  • FIG. 4E is a western blot image showing that effects of ManN are reversible.
  • FIG. 4F is a western blot image showing reduction of molecular mass of VEGFR2, Neuropilin- 1 , CD31 and c-met in HUVEC following ManN treatment at various concentrations.
  • FIG. 4G is a western blot image showing reduction of molecular mass of VEGFR2, (31 integrin and bFGFRl in hDMVECs by ManN at various concentrations. [3-actin served as the loading control.
  • FIGS. 5A to 5D represent examples showing that ManN specifically induces expression of unfolded protein response (UPR) responsive proteins.
  • FIG. 5A is a western blot image. BCECs were grown in Growth Media (GM) until ⁇ 80 % confluency. Media were changed to growth factor-free media containing 10% BCS in the presence or absence of 40 or 400 pM of ManN or mannose for various times. At the end of each incubation, cell lysates were collected, proteins were separated on 4-12% Bis-Tris gel for western blot analysis.
  • FIG. 5B is a western blot image for cells treated with various concentrations of ManN, mannose, 5 ng/ml VEGF or a combination of ManN and VEGF for 24 hours.
  • FIG. 5C is a western blot image showing that 4-PBA, but not TUDCA, could effectively block the induction of CHOP in BCECs, accompanied by a restoration of expression of transcription factor ATF-6 upon 400 pM ManN treatment.
  • BCECs were pre-treated with 2 mM 4-PBA or 500 pM TUDCA, two chemical chaperons. Sixteen hours later, cells were switched to growth factor-free media for 4 hours in the presence of ManN.
  • GM Growth Media.
  • FIGS. 6A to 6J are charts showing effects of ManN on non-endothelial cells of bovine, mouse or human origin.
  • ManN did not promote growth of Calu6 (FIG. 6A), A673 (FIG. 6B), U87MG (FIG. 6C) and 4T1 (FIG. 6D) tumor cells.
  • 10 % FBS was used as positive control for Calu6 and A673
  • 10 ng/ml bFGF and 1 pg/ml human apo-transferrin were used as positive controls for U87MG and 4T1, respectively.
  • no increases in proliferation were induced by ManN on AML 12 (FIG. 6E), bovine pituitary cells (FIG.
  • FIGS. 6F NIH3T3 cells
  • FIG. 6G NIH3T3 cells
  • human RPEs FIG. 6H
  • human dermal fibroblasts FIG. 61
  • human keratinocytes FIG. 6J
  • 6A to 6J are representative western blot analyses showing dose-dependent effects of ManN and mannose at 400 pM (2,4) and 2 mM (3,5) on bFGFRl or [31 integrin (for 4T1, AML12, NIH3T3 cells, human skeletal muscle cells, human dermal fibroblasts and human keratinocytes) molecular mass compared to the untreated control (1).
  • [3-actin served as loading control.
  • FIGS. 7A to 7F represent examples showing effects of protein glycosylation inhibitors on BCEC proliferation.
  • FIG. 7A includes images of samples showing dosedependent stimulation of BCEC proliferation by various inhibitors of glycosylation. Inhibitors were added at concentrations ranging from 0.01 to 100 pM for 3 days, with or without 5 ng/ml VEGF. At the end of the experiment, cells were fixed and stained with crystal violet. A representative experiment is shown. Kifunesine (Kif), an ERa-l,2-mannosidase I and Golgi a-mannosidase I inhibitor; Castanospermine (Cas), an a-glucosidase inhibitor. Cell-covered areas in various treatment groups were quantified by ImageJ software.
  • FIG. 1A includes images of samples showing dosedependent stimulation of BCEC proliferation by various inhibitors of glycosylation. Inhibitors were added at concentrations ranging from 0.01 to 100 pM for 3 days, with or without 5 ng/ml VEGF. At the end of
  • FIG. 7C includes western blot images showing that both inhibitors reduced VEGFR2 molecular mass and induced Bip expression in a dose-dependent fashion as assessed by western blot analysis. Proteins from total cell lysates were separated using 3-8 % Tris-Acetate gel. BCECs were treated with various inhibitors for 24 hours. Quantification of western blots was done by densitometry. [3- actin was the loading control.
  • FIG. 7C includes western blot images showing that both inhibitors reduced VEGFR2 molecular mass and induced Bip expression in a dose-dependent fashion as assessed by western blot analysis. Proteins from total cell lysates were separated using 3-8 % Tris-Acetate gel. BCECs were treated with various inhibitors for 24 hours. Quantification of western blots was done by den
  • FIG. 7E is a western blot image showing activation of AKT and JNK in BCECs by glycosylation inhibitors at 40 pM and VEGF at 10 ng/ml. However, Cas did not activate ERK. Quantification of phosphorylated AKT, JNK and ERK was done by densitometry analysis relative to total protein.
  • FIG. 7E is a western blot image showing activation of AKT and JNK in BCECs by glycosylation inhibitors at 40 pM and VEGF at 10 ng/ml. However, Cas did not activate ERK. Quantification of phosphorylated AKT, JNK and ERK was done by
  • FIGS. 8A to 8D represent examples showing topical application of ManN and VEGF stimulated angiogenesis and accelerates wound healing in mice.
  • FIG. 8A is a chart showing effect of ManN on wounds. Wounds were made on the dorsal skin of mice by 6 mm punch. VEGF and ManN each was administered daily at 20 pg per wound in 25 pl PBS for the first 4 days, with PBS as control. A 10-day wound healing study with 5 mice in each group. Wound closure rate (%) was quantified by Image J software in two independent studies. Asterisks indicated a significant difference compared with the control at each time point.
  • FIG. 8B includes images resulting from a 4-day wound healing study with images of the wound healing process at day 1, day 2 and day 4.
  • FIGS. 9 A to 9D represent examples showing ManN accelerates blood perfusion recovery in a mouse ischemic hindlimb model.
  • FIG. 9A includes images resulting from serial laser Doppler analysis of blood perfusion in hindlimbs of ManN-treated, Kif-treated and control mice. Different colors were used to indicate blood perfusion in the ischemic limb (ligated; left side) to nonischemic limb (sham; right side). Representative images at week 0 and week 1 are shown.
  • FIG. 9C includes images of sample tissue.
  • FIGS. 10A and 10B represent examples showing ManN promotes retinal neovascularization in mice.
  • FIGS. 11 A to 1 ID represent examples showing that ManN, but not structurally related molecules, stimulates endothelial cell proliferation.
  • FIG. 11A is a chart showing additive effects of ManN and bFGF on BCEC proliferation. Bell-shaped effects of ManN on BCEC proliferation. BCECs were treated with ManN ranging from 0.4-400 pM for 5-6 days, with or without 20 ng/ml bFGF. At the end of the experiment, proliferation was quantified using AlamarBlue.
  • FIG. 1 IB is a chart showing that additive effects of VEGF and ManN on BCEC proliferation are dependent on glycolysis pathway. Proliferation assays were carried out in low glucose DMEM media without growth factors or in DMEM media without glucose and pyruvate.
  • the present disclosure provides pharmaceutical compositions and methods for treating an ischemic condition in a subject, including administering to the subject in need thereof an effective amount of hexosamine D-mannosamine (ManN). Because ManN is converted to ManN-6-phosphate (ManN-6p) in vivo, the present disclosure provides for the use of such metabolic precursors and derivatives.
  • ManN hexosamine D-mannosamine
  • ManN-6p ManN-6-phosphate
  • ManN is a hexosamine with an ability to inhibit protein post translational modifications, activate stress pathways and show additivity with VEGF in promoting endothelia; cell (EC) proliferation and angiogenesis. Effects of ManN on ECs and angiogenesis have not been previously reported. Without being bound by theory, using well-known glycosylation inhibitors together with ManN may result in a link between changes in glycosylation patterns in mammalian ECs and angiogenesis. The effects of ManN on endothelial cells may be independent of VEGFR2 activation.
  • ManN was discovered in the 1960s as a bacterial wall component [15], and accounts for 5-10% of capsular polysaccharides [45], The related N-acetyl mannosamine is thought to be an intermediate in the biosynthesis of sialic acids [46], Over the years, multiple effects of ManN on enzymes, growth factor-mediated signaling, protein stability and cell viability were documented [47-50], Most of these effects were not unique to ManN and could be elicited by other hexosamines.
  • ManN was reported to have antitumor properties [47], to stimulate osteogenic differentiation [48, 51] and to protect articular cartilage [49], More recently, ManN was used as an intermediate in modifying various molecules/nanoparticles [52] and in the synthesis of nonnatural ManNAc analogs for the expression of thiols on cell-surface sialic acids to facilitate high-throughput screening [53], However, to date no effects of ManN on ECs have been described.
  • ManN had been previously reported to affect formation of lipid-linked oligosaccharides (LLO) in MDCK cells possibly by inhibiting the a-l,2-mannosyl transferases [24].
  • major oligosaccharides associated with the dolichol were Man5GlcNAc2 and Man6GlcNAc2 rather than Glc3Man9GlcNAc2 which was normally found in MDCK cells.
  • ManN was reported to change protein GPI biosynthesis and hybrid glycans production in the ER [54-56], However, none of the angiogenic-related proteins previously examined are GPI-anchored.
  • Man-9 could be a direct result of inhibiting LLO donor synthesis, i.e. Glc3Man9GlcNAc20PP-Dol formation, which is then transferred from the dolichol donor onto the polypeptide.
  • Man-5 may be significantly increased over 24 hours by treating cells with 40 pM ManN.
  • ManN may be not affect a-mannosidases in the ER.
  • PI3K-AKT PI3K-AKT
  • PLCy-ERK PI3K-AKT
  • p38 Activation of PI3K-AKT, PLCy-ERK and p38 is associated with VEGFR2- mediated EC survival, proliferation and migration.
  • Other cellular metabolic stress sensors such as AMPK (AMP -activated protein kinase), could also confer stress adaptation and promote EC survival via eNOS [57]
  • AMPK AMP -activated protein kinase
  • ACC activation is a general phenomenon for hexosamines and mannose.
  • activation of the JNK/c-Jun and UPR pathways in BCECs is unique to ManN as well as the glycosylation inhibitors. Glycosylation is required for correct protein folding in the ER [26] .
  • a link between LLO inhibition and activation of UPR has been reported [58], In fact, notwithstanding the complexity of ManN actions, LLO inhibition, followed by UPR
  • ECs are able to cope with acute/minor ER stress resulting from glycosylation inhibition by activating the UPR pathway.
  • UPR detects misfolded proteins accumulated in the ER and initiates a response to maintain cellular homeostasis via induction of Bip, a major ER chaperon protein [29], BiP binds to hydrophobic patches exposed on nascent or incompletely folded proteins that are often non-glycosylated.
  • ManN exhibits a strong induction of Bip expression relative to hexosamines. Similar effects on stress pathway activation may result from glycosylation inhibitors Kif and Cas.
  • Glycosylation inhibition is thought to be a new pharmacological strategy targeting metabolic pathways essential for excessive angiogenesis in various pathological conditions, and glycosylation inhibitors are expected to have anti-angiogenic and anti- metastatic properties [10, 59, 60], Glycosylation has been shown to be involved in cellular stress response and compensatory angiogenesis in response to VEGF-VEGFR2 signaling blockade [61], Stress-induced O-GlcNAcylation was previously reported to promote survival in response to DNA damage, ER stress, glucose deprivation and hypoxia in a variety of cell types [62], Without being bound by theory, glycosylation inhibition may be linked to angiogenesis promotion, and inhibiting glycosylation within the tumor microenvironment may result in stimulation rather than suppression of tumor angiogenesis.
  • ManN may be used to promote angiogenesis in a mouse skin injury model, accompanied by accelerated wound closure.
  • ManN may be used to stimulate angiogenesis and blood flow recovery in ischemic hindlimbs of mice.
  • Combinations of ManN, or other glycosylation inhibitors, with VEGF-A, may have advantages over monotherapy for the treatment of ischemic disorders. A lack of direct permeability -enhancing effects of ManN may result in less edematous tissues.
  • damage to lung endothelium is a central pathogenic event in the respiratory failure associated with a variety of infections, including SARS-CoV-2 [65], An endothelial cell mitogen like ManN, devoid of permeabilizing effects, may help protect and stabilize blood vessels and thus limit tissue damage.
  • intravitreal administration of ManN may be used to enhance retinal neovascularization, for example, in therapeutic applications in ocular diseases. 10-15% of patients with intermediate AMD progress to the neovascular form, while the remaining patients may develop geographic atrophy (GA) [1], Previous studies have shown that loss of choroid capillaries is frequently detected in GA, which raises the possibility that regeneration/protection of choroid capillaries may be a strategy for GA treatment [66], [0036]
  • the administration is effective to promote endothelial cell proliferation and angiogenesis in the subject.
  • the method further includes administering to the subj ect in need thereof an effective amount of an N-gly cosylation inhibitor.
  • the method further includes administering to the subject in need thereof an effective amount of VEGF.
  • the ischemic condition is caused by a disease or a trauma.
  • the present disclosure provides for treatment of a number of conditions characterized by reduced perfusion, including but not limited to diabetic ulcers, macular degeneration, peripheral arterial disease (PAD), limb ischemia, brain or cerebral ischemia, and coronary ischemia.
  • the administration is intravenous, intraperitoneal, or intravitreal.
  • the present disclosure provides pharmaceutical compositions and methods for inducing angiogenesis in a subject, including administering to the subject in need thereof an effective amount of hexosamine D-mannosamine (ManN).
  • ManN hexosamine D-mannosamine
  • the administration is effective to reduce ischemia in the subject.
  • the ischemia may include brain ischemia.
  • the administration may be effective in preventing, reducing, or treating conditions associated with brain ischemia, such as edema, ischemic stroke, or infarctions.
  • the method further includes administering to the subj ect in need thereof an effective amount of an N-gly cosylation inhibitor.
  • the method further includes administering to the subject in need thereof an effective amount of VEGF.
  • the subject is in need of inducing angiogenesis due to an ischemic condition is caused by a disease or a trauma.
  • the administration is intravenous, intraperitoneal, or intravitreal.
  • the present disclosure provides pharmaceutical compositions and methods for inhibiting protein glycosylation in a cell, including administering to the cell an effective amount of hexosamine D-mannosamine (ManN).
  • ManN hexosamine D-mannosamine
  • the administration is in vivo. In embodiments, the administration is ex vivo. In embodiments, the administration is effective to stimulate EC proliferation and angiogenesis. In embodiments, the administration is effective to activate JNK and an unfolded protein response caused by ER stress.
  • the administration is effective to induce changes in N-gly can and O-glycan profiles.
  • the administration is effective to induce reduction in Man6GlcNAc2 (Man-6), Man-8 and Man-9 in total oligomannose N-gly can content compared to an untreated control, accumulation of Man-5 and Man-7, and to decrease O-gly cosylation following treatment with ManN.
  • compositions and methods for inhibiting angiogenesis including but not limited to methods for treating malignant tumors and intraocular neovascular disorders in a subject, including administering to the subject in need thereof an effective amount of an inhibitor of hexosamine D-mannosamine (ManN) or reducing the amount of ManN available to the subject.
  • ManN hexosamine D-mannosamine
  • the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by,” or any other variation thereof, are intended to encompass a non-exclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components.
  • a pharmaceutical composition, and/or a method that “comprises” a list of elements is not necessarily limited to only those elements (or components or steps), but may include other elements (or components or steps) not expressly listed or inherent to the pharmaceutical composition and/or method.
  • aspects and embodiments of the present disclosure described herein include “consisting” and/or “consisting essentially of’ aspects and embodiments.
  • the transitional phrases “consists of’ and “consisting of’ exclude any element, step, or component not specified.
  • “consists of’ or “consisting of’ used in a claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component).
  • the phrase “consists of’ or “consisting of’ limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.
  • the transitional phrases “consists essentially of’ and “consisting essentially of’ are used to define a pharmaceutical composition, and/or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed subject matter.
  • the term “consisting essentially of’ occupies a middle ground between “comprising” and “consisting of’.
  • the term “and/or” when used in a list of two or more items, means that any one of the listed items can be employed by itself or in combination with any one or more of the listed items.
  • the expression “A and/or B” is intended to mean either or both of A and B, i.e. A alone, B alone or A and B in combination.
  • the expression “A, B and/or C” is intended to mean A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination or A, B, and C in combination.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • Values or ranges may be also be expressed herein as “about,” from “about” one particular value, and/or to “about” another particular value. When such values or ranges are expressed, other embodiments disclosed include the specific value recited, from the one particular value, and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that there are a number of values disclosed therein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. In embodiments, “about” can be used to mean, for example, within 10% of the recited value, within 5% of the recited value, or within 2% of the recited value.
  • composition refers to pharmaceutically acceptable compositions, wherein the composition comprises a pharmaceutically active agent, and in some embodiments further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition may be a combination of pharmaceutically active agents and carriers.
  • combination refers to either a fixed combination in one dosage unit form, or a kit of parts for the combined administration where one or more active compounds and a combination partner (e.g., another drug as explained below, also referred to as “therapeutic agent” or “co-agent”) may be administered independently at the same time or separately within time intervals.
  • a combination partner e.g., another drug as explained below, also referred to as “therapeutic agent” or “co-agent”
  • the combination partners show a cooperative, e.g., synergistic effect.
  • co-administration or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g., a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time.
  • the term “pharmaceutical combination” as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients.
  • the term “fixed combination” means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient simultaneously in the form of a single entity or dosage.
  • the term “non-fixed combination” means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient.
  • cocktail therapy e.g., the administration of three or more active ingredients.
  • the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia, other generally recognized pharmacopoeia in addition to other formulations that are safe for use in animals, and more particularly in humans and/or non-human mammals.
  • the term “pharmaceutically acceptable carrier” refers to an excipient, diluent, preservative, solubilizer, emulsifier, adjuvant, and/or vehicle with which demethylation compound(s), is administered.
  • Such carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents.
  • Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be a carrier.
  • Methods for producing compositions in combination with carriers are known to those of skill in the art.
  • the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • terapéuticaally effective amount refers to an amount of a pharmaceutically active compound(s) that is sufficient to treat or ameliorate, or in some manner reduce the symptoms associated with diseases and medical conditions.
  • the method is sufficiently effective to treat or ameliorate, or in some manner reduce the symptoms associated with diseases or conditions.
  • an effective amount in reference to diseases is that amount which is sufficient to block or prevent onset; or if disease pathology has begun, to palliate, ameliorate, stabilize, reverse or slow progression of the disease, or otherwise reduce pathological consequences of the disease.
  • an effective amount may be given in single or divided doses.
  • the terms “treat,” “treatment,” or “treating” embraces at least an amelioration of the symptoms associated with diseases in the patient, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. a symptom associated with the disease or condition being treated.
  • treatment also includes situations where the disease, disorder, or pathological condition, or at least symptoms associated therewith, are completely inhibited (e.g. prevented from happening) or stopped (e.g. terminated) such that the patient no longer suffers from the condition, or at least the symptoms that characterize the condition.
  • the terms “prevent,” “preventing” and “prevention” refer to the prevention of the onset, recurrence or spread of a disease or disorder, or of one or more symptoms thereof.
  • the terms refer to the treatment with or administration of a compound or dosage form provided herein, with or without one or more other additional active agent(s), prior to the onset of symptoms, particularly to subjects at risk of disease or disorders provided herein.
  • the terms encompass the inhibition or reduction of a symptom of the particular disease.
  • subjects with familial history of a disease are potential candidates for preventive regimens.
  • subjects who have a history of recurring symptoms are also potential candidates for prevention.
  • the term “prevention” may be interchangeably used with the term “prophylactic treatment.”
  • a prophy tactically effective amount of a compound is an amount sufficient to prevent a disease or disorder, or prevent its recurrence.
  • a prophylactically effective amount of a compound means an amount of therapeutic agent, alone or in combination with one or more other agent(s), which provides a prophylactic benefit in the prevention of the disease.
  • prophylactically effective amount can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.
  • a compound described herein is intended to encompass all possible stereoisomers, unless a particular stereochemistry is specified.
  • structural isomers of a compound are interconvertible via a low energy barrier, the compound may exist as a single tautomer or a mixture of tautomers. This can take the form of proton tautomerism; or so-called valence tautomerism in the compound, e.g., that contain an aromatic moiety.
  • derivative refers to a chemical substance related structurally to another substance, or a chemical substance that can be made from another substance (i.e., the substance it is derived from), e.g., through chemical or enzymatic modification.
  • pharmaceutically acceptable salt refers to acid addition salts or base addition salts of the compounds, such as the multi-drug conjugates, in the present disclosure.
  • a pharmaceutically acceptable salt is any salt which retains the activity of the parent agent or compound and does not impart any deleterious or undesirable effect on a subject to whom it is administered and in the context in which it is administered.
  • Pharmaceutically acceptable salts may be derived from amino acids including, but not limited to, cysteine.
  • a “pharmaceutically acceptable salt” is intended to mean a salt of a free acid or base of an agent or compound represented herein that is non-toxic, biologically tolerable, or otherwise biologically suitable for administration to the subject. See, generally, Berge, et al., J. Pharm. Sci., 1977, 66, 1 -19.
  • Preferred pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of subjects without undue toxicity, irritation, or allergic response.
  • An agent or compound described herein may possess a sufficiently acidic group, a sufficiently basic group, both types of functional groups, or more than one of each type, and accordingly react with a number of inorganic or organic bases, and inorganic and organic acids, to form a pharmaceutically acceptable salt.
  • Examples of pharmaceutically acceptable salts include sulfates, pyrosul fates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen-phosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne- 1,4-dioates, hexyne-1 ,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, methylsulfonates, propylsulfon
  • hexosamine mannosamine (2-Amino- 2-deoxy-D-mannose or ManN hereafter) inhibits protein glycosylation and yet stimulates EC proliferation in vitro.
  • ManN is an EC mitogen and survival factor for bovine and human microvascular EC, with an additivity with VEGF.
  • ManN inhibits glycosylation in ECs and induces significant changes in N-glycan and O-glycan profiles.
  • ManN and two N-glycosylation inhibitors stimulate EC proliferation via both JNK activation and the unfolded protein response caused by ER stress.
  • ManN results in enhanced angiogenesis in a mouse skin injury model. ManN also promotes angiogenesis in a mouse hindlimb ischemia model, with accelerated limb blood flow recovery compared to controls. In addition, intraocular injection of ManN induces retinal neovascularization. Therefore, activation of stress pathways following inhibition of protein glycosylation can promote EC proliferation and angiogenesis and may represent a therapeutic strategy for treatment of ischemic disorders.
  • FIG. 1A and IB A maximal ⁇ 6.5 fold increase in EC-covered surface by ManN at 50 uM alone (FIG. 1 A) or ⁇ 2.5- 3 fold increase in fluorescence units upon AlamarBlue addition (FIG. IB) was obtained when cells were treated with 50 uM ManN and 5ng/ml VEGF, compared to VEGF alone.
  • AlamarBlue detects mitochondria activity as an indication of cell viability which correlates with cell number at certain ranges [16], The effects of ManN had a bell-shaped dose-response curve, with inhibition at higher concentrations (FIGS. 1A and IB). Additive effects of ManN in promoting BCEC proliferation were observed also with bFGF (FIG. 11 A) and in bovine retinal EC (BRECs) (FIGS. 11C and 11D).
  • hexosamines galactosamine, glucosamine and their N-acetyl derivatives
  • ManN ManN in the BCEC proliferation assay.
  • none of these hexosamines had significant stimulatory effects (FIG. ID).
  • Several structurally related molecules such as D-isoglucosamine (fructosamine), meglumine, muramic acid, N-Acetyl- neuraminic acid (sialic acid present in all mammalian cells), glucose, and mannose were also tested. None of these molecules stimulated BCEC proliferation, with or without VEGF (FIGS. 11C and D).
  • ManN entered and accumulated inside the cells in a concentration-dependent manner. 0.66 nmol of ManN were detected in 1 mg cell lysate when BCECs were treated with 400 uM ManN for 2 hours. Following entry into the cells, ManN, but not mannose, is quickly converted to ManN-6-phosphate (ManN-6p) [17], No incorporation of ManN was detected in N-glycans. Efficient uptake of ManNAc, and mannose has been reported [18, 19],
  • FIG. IE shows that 40 uM ManN or 50 ng/ml of VEGF significantly accelerated BCEC migration and/or proliferation as reflected by more complete closure of the “scratched” area, compared to the control group after 48 hours. Similar to the proliferation assays, additivity was observed when cells were treated with both ManN and VEGF (FIG. IE). In addition, ManN at 40 uM showed a significant additivity with VEGF in promoting BCEC migration (FIG. IF).
  • ERK, AKT, mTOR, CREB, AMPK, ACC and eNOS is not unique to ManN. It has been reported that cross-talk between signaling and metabolic pathways in the vasculature, such as insulin signaling and glucose metabolism in ECs, involves AKT and STAT3 activation. Together, they affect glycolysis, EC sprouting, proliferation and migration [20], The effect of ManN and/or VEGF on activating major signal transduction pathways known to promote proliferation, such as ERK, AKT, mTOR and CREB (cAMP response element binding protein) in BCECs was assessed. ManN activated ERK, AKT, mTOR and CREB at 40 uM.
  • ERK, AKT and CREB Stimulation of ERK, AKT and CREB was rapid and occurred within 10-30 minutes after adding ManN (FIGS. 2A to 2C). Further, an enhancement in activation of ERK, AKT and CREB was observed when both ManN and VEGF were present compared to ManN or VEGF alone (FIGS. 2A and 2B). The effect of ManN on activating the ACC (Acetyl-CoA carboxylase)/eNOS (endothelial nitric oxide synthase 3) pathway was assessed.
  • ACC Alcohol-CoA carboxylase
  • eNOS endothelial nitric oxide synthase 3
  • AMPK AMP-activated protein kinase
  • NO nitric oxide
  • JNK/c-jun As a signal transduction pathway uniquely activated by ManN, among hexosamines.
  • Western blot analysis revealed that, among three MAPK family members (ERK, p38, JNK), JNK was specifically activated by ManN.
  • JNK and its downstream c-Jun were significantly activated by ManN in a dose-dependent manner, but not by mannose (FIGS. 3 A and 3B).
  • Treatment of BCECs with the JNK specific inhibitor SP600125 (5 uM) abolished the effects of ManN on BCEC proliferation (FIG. 3C).
  • ManN affects protein glycosylation in endothelial cells.
  • the additivity of ManN with VEGF could potentially occur at transcriptional and/or translational level or through signal transduction pathways mediated by VEGF-VEGFR2.
  • VEGF-VEGFR2 VEGF-VEGFR2
  • FIGS. 5A and 5B FIGS. 7C and 7E
  • Biotinylation studies showed no changes in the amount of VEGFR2 on cell surface.
  • VEGFR2 phosphorylation in response to VEGF was decreased in ManN pre-treated cells, suggesting that VEGFR2 activation was hampered, rather than enhanced in BCECs. No ligand-independent VEGFR2 activation occurred after ManN addition in BCECs. The same was true also for HUVECs (SFIG. 10a) and hDMVECs.
  • ManN may affect VEGFR2 post translational modification
  • cells were treated with ManN in the presence of one of four monosaccharides (mannose, glucose, galactose, or fucose) at a maximum 1:10 molar ratio.
  • monosaccharides are known to be important in protein N-glycosylation.
  • mannose could dose-dependently block ManN effects on VEGFR2 molecular mass as well as on BCEC proliferation (FIGS. 4C and 4D).
  • the effects of mannose may not be limited to prevention of entry of ManN into cells via the same transporter(s) since the effects were seen when BCECs were first treated with ManN for 2 hours to ensure its successful cellular uptake.
  • Glucose, but not galactose or fucose had similar effects as mannose.
  • N- glycosylation is a complex process, dependent on multiple enzymes that act sequentially on glycoproteins to generate hybrid and high-mannose glycan structures as they transit through the secretory pathway, from ER to Golgi apparatus [22], It plays an important role in the determination of the fate of newly synthesized glycoproteins in the ER, their correct folding, cellular destination and proper function.
  • a-mannosidase from Jack Bean is a broad-specificity exoglycosidase that catalyzes the hydrolysis of terminal, non-reducing al -2, al -3, and al -6 linked mannose residues from oligosaccharides in both organelles, and controls conversion of high mannose to complex N-glycans, the final hydrolytic step in the N-glycan maturation pathway.
  • This enzyme has been used to screen for potential N-glycosylation inhibitors [23], ManN, but not other hexosamines or their derivatives, showed inhibitory activity at 400 uM, which is considerably higher than the effective mitogenic concentrations in BCECs. No effect of ManN up to 2 mM on a- or [3-glucosidases was detected.
  • N-linked glycans from BCECs were isolated by enzymatic cleavage, followed by purification and characterization using MALDI-TOF-MS.
  • Treatment with 40 uM ManN resulted in a significant time-dependent reduction in Man6GlcNAc2 (Man-6), Man-8 and Man- 9 in total oligomannose N-glycan content compared to the untreated control, whereas a significant early phase accumulation of Man-5 and Man-7 was observed after ManN treatment.
  • ManN has been previously shown to inhibit Lipid-linked oligosaccharide (LLO) synthesis, to change protein GPI biosynthesis and hybrid glycan production as well as to incorporate into the glycans in MDCK cells [24], Accumulation of Man-5 over time suggested that inhibition of mannosidase is unlikely the mechanism of pro-angiogenic activity in BCECs.
  • LLO Lipid-linked oligosaccharide
  • the monosaccharide content was measured to profile the composition of complex N-glycans.
  • a significant decrease in fucose (8 hours), mannose (12 hours), galactose (24 hours) and Neu5Ac (8 hours and 24 hours) were found in ManN-treated cells compared to the untreated control cells, consistent with the inhibitory activity of ManN on overall protein N-glycosylation.
  • O-glycan modification is another form of post translational modification of proteins, where a serine or threonine residue is covalently linked with a GalNAc residue [22],
  • the GalNAc residue can be further modified by several glycosyl-transferases acting in a sequential manner to extend the glycan chain, either branched or linearly, according to substrate specificity.
  • the ppGalNAcT polypeptidyl GalNAc transferase catalyzes the transfer of a a- GalNAc from UDP-GalNAc to Ser or Thr residue of a glycoprotein, producing the Tn antigen.
  • the Tn antigen When the Tn antigen is generated, it can have three different fates: (i) it can be sialylated on C6 by the enzyme ST6GalNAcT; (ii) it can be substituted on C3 or C6 by a [3-GlcNAc which gives rise to core-3 or core-6, respectively; or (iii) it can be galactosylated on C3 by the CIGalTl in order to form core-1 which can also be sialylated to produce mono- or di-sialyl Core-1 O-glycan [22],
  • O-glycan analysis was conducted in BCEC lysates by MALDI-Tof mass spectrometry. Due to unavailability of a unique enzyme that cleaves all different forms of O- glycan, a reductive beta-elimination was performed to have an understanding of the O-glycan backbone [25], To protect from de-sialylation during mass spectral data acquisition, permethylation was performed prior to MALDI-Tof/Tof mass analysis [25], An overall decrease in O-glycosylation following treatment with 40 uM ManN.
  • ATF-6 is cleaved liberating a 50 kDa amino-terminal fragment that translocates to the nucleus which activates transcription of ER chaperones and ER-associated degradation components such as Bip and CHOP upon accumulation of improperly folded proteins in the ER, [28], Pre-treating cells with 1 mM 4-PBA for 4 hours could effectively reverse the bell-shaped activity of ManN on BCEC proliferation in the absence or presence of VEGF. Additivity between ManN and VEGF was largely abolished (FIG. 5D).
  • BRECs Fig. 1C
  • hRMVECs Fig. 1C
  • HUVECs Fig. 6
  • hDMVECs hDMVECs
  • no proliferative effects by ManN were observed at pM to mM concentrations, alone or in combination with other growth stimulators (FIG. 6), although efficient ManN uptake and comparable levels of free ManN were detected in all cell types.
  • FIG. 7E illustrates a dosedependent induction of Bip expression when growing BCECs were switched to media without growth factors for 24 hours in the presence of Kif or Cas at concentrations which promoted cell proliferation.
  • the combination had a significant faster wound closure starting from day 2 (FIG. 8B).
  • an average closure of the wound was 81.5%, 75.6%, 66.9% and 29.8% in PBS-, ManN-, VEGF- and combination-treated group, respectively.
  • Small vessel numbers were quantified around the wound area at day 4.
  • a significant increase in CD31-positive vessels was found in the combination group compared to PBS control, VEGF or ManN alone (FIGS. 8C and 8D).
  • ManN in combination with VEGF, promotes angiogenesis in a skin injury model.
  • wound closure takes place rapidly, without any treatment.
  • ManN The stability of ManN was assessed in wound fluid contaminated by bacteria, a common feature of wounds. ManN was added to freshly collected wound fluid from a mouse model of skin infection with Staphylococcus aureus, a prevalent cause of skin and soft tissue infections in humans [39], No significant loss of free ManN was detected following incubation with such wound fluid for up to 24 hours at 37 °C. Thus, ManN may be useful for treatment of infected wounds, possibly in combination with anti-microbials or other agents.
  • VEGF vascular endothelial growth factor
  • the perfusion ratio in LLO-fed group indicated a blood flow recovery of -25%, a value that is in good agreement with published data with the same type of lesion, in the same strain of mice [40, 44],
  • the blood flow recovery in ManN and Kif-treated group was about 40% and 47%, respectively, which demonstrated an accelerated recovery rate of blood flow compared to H2O-treated mice (FIGS. 9A and 9B).
  • the blood perfusion ratio continued increasing to -50% of sham-treatment limbs in 3 weeks after ManN and Kif treatment and was significantly higher than the control group (FIGS. 9A and 9B).
  • the ischemic hindlimbs of ManN- treated and Kif-treated group showed an increased blood vessel density compared to the control group, as assessed by CD31 immunostaining of the surrounding muscle tissue 3 weeks postligation.
  • blood vessel densities were respectively 2.3 and 1.8 times higher in ManN and Kif-treated groups (FIGS. 9C and 9D).
  • ManN plasma levels Following oral administration, there was a relatively rapid decline in ManN plasma levels. Plasma free ManN levels reached a peak level of -100 nmol/ml plasma at 1 hr. After 3 hours, only about half of that amount was detectable. 2 hours after oral feeding of 20% ManN, muscle samples were taken from the ischemic legs. A significant amount of ManN reached the ischemic legs, with 0.17+/-0.18 nmol/mg protein of free ManN and 0.91+/-0.24 nmol/mg protein of ManN-6p. At least in BCECs, ManN effects on protein mass lasted for at least 8 hours in the absence of exogenous ManN (FIG. 4E), indicating that even a relatively brief exposure may be adequate to elicit pharmacological effects.
  • Kif was also tested in this model because it is a water- soluble inhibitor and its mechanism of glycosylation inhibition is well established [35], In addition, it shares with ManN the ability to activate ERK, AKT and stress pathways in BCEC (FIG. 7E).
  • MSMLS Mass Spectrometry Metabolite Library of Standards
  • IROA TECHNOLOGIES Bolton, MA; now Sigma
  • MSMLS Mass Spectrometry Metabolite Library of Standards
  • D-Mannosamine hydrochloride was obtained from Sigma (M4670) or Spectrum Chemical MFG Corp (M3220).
  • 1-Amino-l-deoxy-D-Fructose hydrochloride (D- isoglucosamine) (803278), D-(+)-Galactosamine (1287722), D-(+)-Glucosamine (1294207), N-acetyl-Mannosamine (A8176), N-acetyl-galactosamine (A2795), N-Acetyl-Glucosamine (A8625), Meglumine (M9179), Muramic acid (M2503), N-Acetylneuraminic acid (A2388), D- (+)-Glucose (D9434), D-(+)-Mannose (1375182), Meglumine (M9179), Tunicamycin from Streptomyces sp.
  • Antibodies used in the present study were from Cell signaling Technology Inc. (Danvers, MA) unless otherwise specified. Total: VEGFR2 (2479), ERK (4695), p38 (9212), JNK (9252), mTOR (2983), AKT (4691), CREB (9104), CHOP (2895), ACC (3676), ATF-6 (65880), Bip (3183), AMPKD (5832), FGFR1 (9740), eNOS (9586), VE-Cadherin (2500), c- Met (3127 or 3148), Neuropilin (3725), CD31 (3528), c- Jun (9165).
  • VEGFR2 (Tyrl l75, 2478 or 3770), ERK1/2 (Thr202/Tyr204, 4376), p38 (Thrl80/Tyrl82, 4511), JNK (Thrl83/Tyrl85, 9251), mTOR (Ser2448, 5536), AKT (Ser473, 4060), CREB (Serl33, 9191), ACC (Ser79, 3661), eNOS (Seri 177, 9571), AMPKa (Thrl72, 50081), c-Jun (Ser73, 9164), pi integrin (4706 & 34971), av integrin (4711), JNK1 (3708), JNK2 (4672), JNK3 (2305). Anti- -actin was from Sigma.
  • Bovine retinal microvascular endothelial cells BRECs, #BRMVEC-3
  • bovine choroidal microvascular endothelial cells BCECs, #BCME-4
  • VEC Technologies Renssellaer, NY
  • Human retinal microvascular endothelial cells (passage ⁇ 15) was from Cell Systems Corporation (Kirkland, WA). They were grown on 0.1% Gelatin-coated plates in Medium 131 containing 5% fetal bovine serum, hydrocortisone (1 pg/ml), human fibroblast growth factor (3 ng/ml), heparin (10 pg/ml), human epidermal growth factor (1 ng/ml) and dibutyryl cyclic AMP (0.08 M) (MVGS, S 005-25, Gibco Invitrogen).
  • the human RPE cell line ARPE-19 was from the ATCC.
  • RtEGM media (Clonetics) containing 2% FBS, L-glutamine, human bFGF, GA-1000). Once cells attached to plates, serum free RtEGM media was used to maintain the culture for best result.
  • ARPE-19 was obtained from ATCC (CRL-2302) and cultured according to company’s instruction.
  • NIH3T3 cells were obtained from ATCC (CRL-1658). Human adult dermal MVECs (CC- 2543) were cultured in EGM-2MV (CC-4147, Lonza).
  • Keratinocytes were cultured in dermal cell basal media (PCS-200-030) plus keratinocyte growth kit (PCS- 200-040).
  • Human primary dermal fibroblasts (ATCC PCS-201-012) were cultured in fibroblast basal medium (ATCC, PCS-201-030) plus growth kit (ATCC, PCS-201-040).
  • Growth stimulators used in the assay included human EGF (R&D systems, 236-EG), murine TGFP (R&D systems, 410-MT), KGF (Sigma, K1757), or 10% FBS growth media.
  • 4T1 cells were obtained from the ATCC (CRL-2539) and cultured in RPMI-1640 with 10% FBS (Omega Scientific, Tarzana, CA) and antibiotics.
  • A673 (CRL-1598), A549 (CCL-185), U87MG(HTB- 14) cells were from ATCC and cultured in high glucose DMEM containing 10% FBS.
  • FBS (S12550) was purchased from R&D systems.
  • BCS (SH30073.03) was obtained from Hy clone. All cell lines used in the study are negative for mycoplasma contamination by various vendors.
  • a hypoxic condition cells were placed in a hypoxia incubator with a mixture of gas consisting of 1% O2, 5% CO2 and 94% N2.
  • a mixture of gas consisting of 1% O2, 5% CO2 and 94% N2.
  • untreated and VEGF -treated (10/ng/ml) wells were included to monitor plate-to-plate variations.
  • 20% methanol or 0.05% DMSO served as negative controls.
  • 0.05% DMSO served as negative controls when Cas was tested in these cells.
  • Human RMVECs and human adult DMVECs were split into Gelatin-coated 96 wells (2000 cells per well) in low glucose DMEM containing 10% FBS. 1200 cells/well was set up for proliferation assay in low glucose media containing 0.5% FBS. Data was collected at day 4 or 5.
  • HUVEC (p7- 10) were grown on gelatin-coated plate until it reached 70-80% confluency.
  • Proliferation assays with fibroblasts were done in low-glucose DMEM containing 1% FBS, with or without 10 ng/ml bFGF, or 100 ng/ml human EGF and the assay was ended at day 3.
  • ARPE-19 cells were gently lifted with 0.025% trypsin and plated in RtEGM media (Clonetics) containing 2% FBS, L-glutamine, human bFGF and GA-1000. Once cells were attached to plates, serum-free RtEGM media was used to maintain the cultures.
  • For proliferation assays 1500 human RPE cells were plated into 96-well plates in low-glucose DMEM containing 1% FBS.
  • A673, U87MG, Calu6 and AML12 cells were grown until confluent and were then harvested and re-suspended in appropriate assay media.
  • cells were plated at the density of 1000-2000 cells/well in low-glucose DMEM containing 5% FBS or otherwise stated.
  • Bovine pituitary cells pituitary folliculostellate cells
  • human epidermal keratinocytes, human DMVECs and human dermal fibroblast cells 1000 cells/well were plated in low-glucose DMEM containing 1% FBS with or without various growth factors.
  • the assay was ended at day 3 for bovine pituitary cells and at day 4 for all the other cell types.
  • 4T1 1000 cells were plated in RPMI-1640 with 2% basement membrane extract (BME) and 2% FBS on BME-coated 96-wells and treated 4 hrs later [68], Four days later, tumor cell growth was measured by the MTS assay (Promega, Madison, WI), a colorimetric assay that measures metabolic activity of viable cells.
  • Recombinant human transferrin was obtained from EMD Millipore (Temecula, CA).
  • Recombinant mouse apotransferrin was obtained from Sigma.
  • BCECs were plated in 6-well culture plates at a density of 1.5X10 5 cells/well and cultured overnight. 2 ml of antibiotics-free culture medium was used to replace the old medium.
  • siRNAs including siNegative (Ambion, AM4611), siRNA against JNK1#2 (Invitrogen, NM_001192974.2_siRNA_266), JNK1#4 (Invitrogen, NM_001192974.2_siRNA_485), siRNA against JNK2#2 (Invitrogen,
  • RNAiMAX reagent (ThermoFisher Scientific, 13778150) in Opti-MEM I Reduced Serum Medium (Gibco, 31985062) according to manufacturer’s instructions. Briefly, a mix containing 25 pmol of siRNA, 7.5 pl of RNAiMAX reagent and 125 pl of Opti-MEM medium was used to transfect cells in each well, to a final siRNA concentration of 12.5 nM. A mix of RNAiMAX and Opti-MEM was used as no siRNA control. Cells were incubated with siRNAs. 8 hrs later, the siRNA-containing medium was replaced with fresh medium. 24 and/or 48 hrs after transfection with siRNAs, cells were used for proliferation assays and protein extraction.
  • Glycerol-free PNGase F was obtained from New England Biolabs (Ipswich, MA). Briefly, BCECs were lysed with NP-40 containing proteinase inhibitors (Thermo Scientific, Waltham, MA). Lysates were cleared at 4°C at 5000 g for 25 mins. Total protein content was measured using Pierce BCA protein assay kit (ThermoScientific). 20 mg of protein was mixed with 10X denaturing buffer and H2O to a total volume of 10 ml. Glycoproteins were denatured at 100°C for 10 mins, followed by adding Glycobuffer and PNGase F. The reaction was carried out at 37°C for 2 hr.
  • Cells were allowed to reach -80% confluency in 12-well plates. Cells were pre-treated with ManN, Kif or Cas for various time durations, with or without the subsequent addition of VEGF, with H2O as the solvent control for ManN. At various time points, plates were taken out of the incubator and kept on ice.
  • Cell monolayers were first washed once with ice-cold PBS before lysis with 250 pl of Pierce RIPA buffer (ThermoFisher Scientific, Rockford, IL) or use 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 10% Glycerol, 1% NP-40 containing protease/Phosphatase inhibitor cocktail (100X) (Cell signaling, #5872). Lysates were collected and mixed with 4X Bolt LDS Sample Buffer (Novex, Carlsbad, CA) in the presence of Halt protease inhibitors and phosphatase inhibitor cocktail (ThermoFisher scientific, #NP0007).
  • HUVECs (passage 6-8) were plated in EBM-2 basal medium (Lonza) with 0.2% FBS. Following overnight culture, cells were serum-starved in EBM-2 medium for 4 hrs prior to treatment with 50 ng/ml of VEGF165 or vehicle controls for various lengths. Equal amounts of protein lysates were analyzed by SDS-PAGE and blotted with the indicated antibodies.
  • Proteins were transferred using Tris-Glycine buffer with 20% Methanol (Proteonomics grade) (Apex BioResearch Products). Membranes were first incubated with 5% milk in TBST, pH 7.6 (TEKnova, Hollister, CA), followed by blotting with primary and secondary antibodies. ECL anti-rabbit IgG, horseradish peroxidase linked whole antibody from donkey or sheep anti-mouse were obtained from GE Healthcare (UK limited). SuperSignal West Dura Extended Duration substrate was from ThermoFisher Scientific. In some cases, the same PVDF membranes were stripped by 8-min incubation in the Restore Plus Western Blot Stripping Buffer (ThermoFisher Scientific) to show total specific protein expression, followed by second stripping for P-actin expression.
  • HUVECs (passage 6-8) were cultured and serum-starved as described in “Western Blots”. Ten thousand cells in 150 pl of EBM-2 medium were then added to the upper chamber of 8 pm pore size cell culture inserts (Falcon) coated with 0.1% gelatin. The lower compartment was filled with 600 pl EBM-2 medium containing various agents. The plates were incubated at 37°C to allow migration. After 4 hrs, cells were fixed with 4% PFA for 20 min and then stained with crystal violet (Sigma- Aldrich) for 20 min at RT. Migrated cells on the bottom side of the insert membrane were quantified by counting whole area of the insert at 40X magnification. The experiments were carried out in triplicate and repeated three times. BCEC migration was set up similarly, except that wells were coated with FN, cells were suspended in 1% serum media and migration time was 18-24 hrs.
  • BCECs (passage 6-10) and HUVEC (passage 6-8) were used in this assay.
  • Cells were grown until about 80% confluency in 6-well plates, washed twice with PBS and then starved in serum-free DMEM (low Glucose, Hy clone) for 5 hrs before making a “scratch” using 1ml tip.
  • DMEM low Glucose, Hy clone
  • Cell monolayers were briefly washed once with serum-free media, followed by various treatments in media containing 1% FBS. 48 hrs later, the assay was stopped by adding 2ml 4% paraformaldehyde. 20 min later, fixed cells were stained with 1ml Crystal Violet (Sigma). Plates were washed gently under the running tap water and air-dried before taking pictures.
  • N-Glycan monosaccharide, sialic acid, O-glycan analysis
  • BCECs were washed twice with phosphate buffered saline (PBS, Sigma) and harvested by scraping. The cells were pelleted by centrifugation at 300 g for 3 mins and washed once with cold PBS. Cells were homogenized and total protein was measured. All subsequent analysis was based on known protein amount.
  • N-linked glycans were removed from glycoprotein samples using PNGase-F kit (New England BioLabs, P0705S). Briefly, 300 pg of protein sample was reconstituted in 180 pl UltraPure water. 20 pl of 10X denaturing buffer was added and boiled using 100°C water bath for 14 mins.
  • Samples were cooled down to room temperature and centrifuged at 2700 g for 1 min. Subsequently, 50 pl 10X NP-40 was added and samples were kept at room temperature for 30 mins with vortexing at 5 min interval, followed by adding 25 pL of 10X reaction buffer and mixing thoroughly. 5 pl PNGaseF (2500 U) was then added to the samples and mixed gently. Samples were incubated at 37°C for 16 hrs. Released N-glycans were purified using solid phase extraction method.
  • N-glycans were purified by passing the reaction mixture sequentially over pre-conditioned Sep-Pak C18 lee cartridge (Waters) and HyperSep PGC (poly graphitized charcoal) cartridge (25 mg, 1 ml Thermo Scientific). The cartridge was washed with 4 ml of water and the PGC alone was washed with additional 1 ml of water. N-glycans bound to PGC were eluted using 30% acetonitrile containing 0.1% TFAin water. Finally, purified N-glycans were lyophilized and labeled with 2-AB.
  • Sep-Pak C18 lee cartridge Waters
  • HyperSep PGC poly graphitized charcoal
  • Monosaccharide profile was done using Dionex CarboPacTM PAI column (250mm x 4mm; with 50mm x 4mm guard column). An isocratic solvent mixture of 19 mM sodium hydroxide with 0.95 mM sodium acetate was used at a flow rate of 1 ml per minute for 25 mins. Data were acquired using manufacture supplied standard Quad waveform for carbohydrates. All neutral and amino sugars were identified and quantified by comparing with authentic monosaccharide standard mixture consisting of L-fucose, D-galactosamine, D-glucosamine, D-galactose, D-glucose and D-mannose [70],
  • the cell homogenate was filtered through pre-washed 3K filters and the filtrate was dried using speed-vac.
  • the dry sample was reconstituted in 100 pl of ultrapure water and sample with 200 pg equivalent of protein was injected onto HPAEC-PAD.
  • a known amount (1 nmol) of ManN, Glucose, Mannose, and ManN-6P standards were used to quantify the sugars present in the samples. All standards, except ManNH2-6P, were obtained from Sigma-Aldrich.
  • ManNH2-6P was from Omicron Biochemicals, Inc (South Bend, IN). The amounts of monosaccharides present in different cells are presented as nmol/mg of total protein amount. All analyses was performed in a Thermo-Dionex ICS system using a CarboPac-PA-1 column in 100 mM NaOH and 250 mM NaOAc as HPLC running buffer.
  • BCECs were plated in 10 cm cell culture dishes 3 days prior to the cell surface protein isolation. Cells were washed three times with Dulbecco’s PBS with CaCh and MgCh, followed by a 30 min incubation with EZ-Link Sulfo-NHS-SS-Biotin (Pierce, Rockford, IL, USA; 0.5 mg/ml in Dulbecco) on ice. Cells were washed twice with Dulbecco and the nonreacted biotin was blocked with 20 mM glycine for 15 mins.
  • a 100 uM oxidized glutathione (Sigma-Aldrich, St. Louis, MO) was added in the last wash solution.
  • lysis buffer 20% NP-40, 1% Triton X-100, 10% glycerol, 100 uM oxidized glutathione, EDTA free protease inhibitor tablet (Roche, Mannheim, Germany) in PBS was added to the cells. Lysed cell extracts were scraped off the plates and transferred to an Eppendorf tube followed by incubation on ice on a shaker for 30 mins.
  • the cell extracts were incubated with 30 U of DNase (22 °C 50 mins, Roche, Mannheim, Germany) and centrifuged for 20 mins (20,800*g, at 4 °C) to pellet the insoluble material. The protein concentration of the supernatants was determined. Equal amounts of protein ( ⁇ 2 mg) from each extract were used for cell surface protein isolation. The supernatant was pre-cleared using biotin agarose beads (Pierce ImmunoPure Immobilized D-biotin, Thermo Scientific, 20221) and pre-cleared solution was used for the cell surface protein isolation using streptavidin beads.
  • Bovine VEGF-A (Bt03213282), bovine RPL19 (Bt03229687) and bovine specific VEGFR2 (Bt03258877), GLUT1 (Bt03215313) and GLUT4 (Bt03215316).
  • a-mannosidase activity was measured using substrate -nitrophenyl a- mannopyranoside (ImM).
  • Enzyme from Jack Bean (M7257) (final concentration of 0.077 U) was incubated at 37°C in a final volume of 50 pl of 50 mM potassium phosphate buffer, pH 7.5.
  • a-glucosidase was assayed with substrate -nitrophenyl a-glucoside (7 mM).
  • Enzyme from Saccharomyces cerevisiae-type 1 (Sigma, G5003) (final concentration of 0.1 U) was incubated at 37°C in a final volume of 50 pl of PBS, pH 7.5.
  • 0-Glucosidase was assayed with substrate 4-nitrophenyl 0-D-glucopyranoside (Roche).
  • Enzyme from almond (Sigma, G0395) (final concentration of 0.002 U) was incubated at 37°C in a final volume of 50 pl of PBS, pH 7.5 containing 1% SDS. The incubation was stopped by addition of an equal volume of acidbased stop solution (R&D systems, 895032). Enzymatic activity was measured at 405 nm.
  • a- glucosidase from Saccharomyces cerevisiae type I (G5003) with -nitrophenyl a-D- glucopyranoside (Sigma, N1377) as the substrate.
  • ManN was identified and quantified by comparison with monosaccharide standard using Thermo Scientific Chromeleon software. No ManN samples served as negative controls.
  • VEGF vascular endothelial growth factor
  • a sterile neoprene ring (6-mm outer diameter and 4-mm inner diameter), fastened with 5-6 sutures (4-0 nylon) under the influence of Isoflurane was created on the back of the animal in a Class II Biological Safety Cabinet.
  • sterile technique was followed. Buprenorphine was given subcutaneously prior to awakening from anesthesia for anticipated pain. Mice were monitored until fully awake and were housed individually to minimize damage/bi ting/ fighting to the surgical site.
  • Recombinant human VEGF was a gift from Roche-Genentech (Telbermin, recombinant human VEGFies).
  • Treatment agents were prepared in PBS, sterile-filtered and 25 pl solution was applied daily directly to a wound bed for the first 4-5 days under the influence of Isoflurane, followed by daily observation. Wound closure was monitored by regular imaging and the wound area was quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA).
  • Immunostaining was performed as previously described [20], Anti-CD31 (SZ31, rat IgG2a) (Dianova, Warburgstrasse 45, 20354 Hamburg, Germany) was used at 2 pg/ml. Small vessels stained positive for CD31 were counted microscopically on 10 fields (20X) taken around the wound.
  • vascular permeability was assessed using a modified Miles assay[14].
  • Hairless male guinea pigs (Crl: HA-Hrhr/IAF, 75 days old, 450-500 g, Charles River Laboratories) were anesthetized by intraperitoneal (/./?.) administration of xylazine (5 mg/kg) and ketamine (75 mg/kg). The animals then received an intravenous injection (penile vein) of 1 ml of 1% Evans blue dye. After 15 mins, intradermal injections (0.05 ml/per site) of different doses of ManN were administrated into the area of trunk posterior to the shoulder. All reagents were diluted in PBS for intradermal administration.
  • VEGF165 25ng was used as positive control. 30 mins after the intradermal injections, animals were euthanized by i.p. injection of pentobarbital (200 mg/kg). Skin tissues were dissected from the connective tissues and photographed.
  • a skin infection model in the mouse was established [39], Briefly, mid-log phase of Staphylococcus aureus sub-cultured from overnight cultures in Todd Hewitt broth were used in this study. 6-8 weeks old C57BL/6 mice were obtained from Charles River Laboratories. Mice were shaved and depilated by Nair cream before infection. 5xl0 7 CFU of 5. aureus was intradermally injected into the left groins of mice. After 3 days, abscesses were surgically removed and homogenized on ice. Fluid was collected and spun at 14000 rpm. Cleared supernatant was diluted with PBS 1:1 for further use. Animals were housed in clean cages and experimental procedures hereafter were carried out under pathogen-free conditions. The presence of bacteria in the wound fluid was confirmed using Todd Hewitt Broth (THB) plates.
  • TTB Todd Hewitt Broth
  • mice C57BL/6 male mice (6-8 weeks old) were subjected to unilateral hindlimb surgery under anesthesia with ketamine/xylazine cocktail [41, 43], Briefly, the left femoral artery was separated from the vein and nerve, ligated proximally, and excised. The right hindlimb served as control. Blood flow was measured by using a laser Doppler perfusion imager (PeriScan PSI; Perimed). Ischemic and nonischemic limb perfusion was measured before and after surgery and 1, 2 and 3 weeks later. After surgery, mice were randomly allocated to different groups (8 mice for each group). 200 pl of 20% ManN was orally administrated every other day from 3 rd day after surgery.
  • PeriScan PSI Perimed
  • rat anti-mouse antibody (BD Biosciences, CAT# 550274) was diluted 1:100 and incubated overnight at 4 °C. After 4-hour incubation with the Alexa Fluor-488-conjugated anti-rat antibody (Life Technologies, Al 1006), whole mounts were imaged via the 488 nm channel using AIR Confocal STORM super-resolution system (Nikon). Quantification of vascular density in choroids and retina was carried out by Image J. Each experiment was repeated three times with similar results, and each treatment group consists of 5 individual samples.
  • a method of treating an ischemic condition in a subject including administering to the subject in need thereof an effective amount of hexosamine D-mannosamine (ManN).
  • ManN hexosamine D-mannosamine
  • Aspect 2 The method of aspect 1, wherein the administration is effective to promote endothelial cell proliferation and angiogenesis in the subject.
  • Aspect 3 The method of aspects 1 or 2, wherein the method further includes administering to the subject in need thereof an effective amount of an N-glycosylation inhibitor.
  • Aspect 4 The method of any of aspects 1 to 4, wherein the method further includes administering to the subject in need thereof an effective amount of VEGF.
  • Aspect s The method of any of aspects 1 to 5, wherein the ischemic condition is caused by a disease or a trauma.
  • Aspect 6 The method of any of aspects 1 to 6, wherein the administration is intravenous, intraperitoneal, or intravitreal.
  • Aspect 7 A method of inducing angiogensis in a subject, the method including administering to the subject in need thereof an effective amount of hexosamine D- mannosamine (ManN).
  • ManN hexosamine D- mannosamine
  • Aspect 8 The method of aspect 7, wherein the administration is effective to reduce ischemia in the subject.
  • Aspect 9 The method of aspects 7 or 8, wherein the method further includes administering to the subject in need thereof an effective amount of an N-glycosylation inhibitor.
  • Aspect 10 The method of any of aspects 7 to 9, wherein the method further includes administering to the subject in need thereof an effective amount of VEGF.
  • Aspect 11 The method of any of aspects 7 to 10, wherein the subject is in need of inducing angiogenesis is due to an ischemic condition caused by a disease or a trauma.
  • Aspect 12 The method of any of aspects 7 to 11 , wherein the administration is intravenous, intraperitoneal, or intravitreal.
  • a method of inhibiting protein glycosylation in a cell including administering to the cell an effective amount of hexosamine D-mannosamine
  • Aspect 14 The method of aspect 13, wherein the administration is in vivo.
  • Aspect 15 The method of aspects 13 or 14, wherein the administration is ex vivo.
  • Aspect 16 The method of any of aspects 13 to 15, wherein the administration is effective to stimulate EC proliferation and angiogenesis.
  • Aspect 17 The method of any of aspects 13 to 16, wherein the administration is effective to activate JNK and an unfolded protein response caused by ER stress.
  • Aspect 18 The method of any of aspects 13 to 17, wherein the administration is effective to induce changes in N-glycan and O-glycan profiles.
  • Pili, R., et al. The alpha-glucosidase I inhibitor castanospermine alters endothelial cell glycosylation, prevents angiogenesis, and inhibits tumor growth. Cancer Res, 1995. 55(13): p. 2920-6.
  • Estrada-Mondaca, S., L.A. Delgado-Bustos, and O.T. Ramirez, Mannosamine supplementation extends the N-acetylglucosaminylation of recombinant human secreted alkaline phosphatase produced in Trichoplusia ni (cabbage looper) insect cell cultures. Biotechnol Appl Biochem, 2005. 42(Pt 1): p. 25-34.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Vascular Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Immunology (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Organic Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des compositions pour traiter un état ischémique chez un sujet qui peuvent comprendre de l'hexosamine D-mannosamine (ManN). Les procédés peuvent comprendre l'administration au sujet qui en a besoin d'une quantité efficace de ManN. L'administration peut être efficace pour favoriser la prolifération des cellules endothéliales et l'angiogenèse chez le sujet. Le sujet peut avoir besoin d'une angiogenèse induite en raison d'un état ischémique provoqué par une maladie ou un traumatisme. Les compositions destinées à inhiber la glycosylation des protéines dans une cellule peuvent comprendre de la ManN. Les procédés d'inhibition de la glycosylation des protéines dans une cellule peuvent comprendre l'administration à la cellule d'une quantité efficace de ManN.
EP21883794.6A 2020-10-20 2021-10-20 Compositions et procédés de traitement d'états ischémiques Pending EP4232454A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202063094032P 2020-10-20 2020-10-20
PCT/US2021/055809 WO2022087107A1 (fr) 2020-10-20 2021-10-20 Compositions et procédés de traitement d'états ischémiques

Publications (2)

Publication Number Publication Date
EP4232454A1 true EP4232454A1 (fr) 2023-08-30
EP4232454A4 EP4232454A4 (fr) 2024-09-18

Family

ID=81289410

Family Applications (1)

Application Number Title Priority Date Filing Date
EP21883794.6A Pending EP4232454A4 (fr) 2020-10-20 2021-10-20 Compositions et procédés de traitement d'états ischémiques

Country Status (10)

Country Link
US (1) US20230390315A1 (fr)
EP (1) EP4232454A4 (fr)
JP (1) JP2023545826A (fr)
KR (1) KR20230091119A (fr)
CN (1) CN116507340A (fr)
AU (1) AU2021365844A1 (fr)
CA (1) CA3195941A1 (fr)
IL (1) IL302158A (fr)
MX (1) MX2023004525A (fr)
WO (1) WO2022087107A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2025514744A (ja) * 2022-04-20 2025-05-09 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 脳卒中に対する治療薬としてのグリコシル化阻害剤

Family Cites Families (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4290231B2 (ja) * 1997-04-09 2009-07-01 生化学工業株式会社 角膜障害症治癒促進剤
JP3545601B2 (ja) * 1998-05-20 2004-07-21 花王株式会社 ラクトシルセラミド合成酵素阻害剤
WO2000007602A1 (fr) * 1998-08-06 2000-02-17 The Johns Hopkins University School Of Medicine Composes permettant d'alterer les acides sialiques d'une surface cellulaire et leurs methodes d'utilisation
EP1417971A3 (fr) * 1998-09-09 2004-06-30 Scios Inc. Utilisation d' un facteur angiogénique pour le traitement d'angiopathies microvasculaires
US20030105034A1 (en) * 2001-07-05 2003-06-05 Astion Deveopment A/S Pyridine carboxy derivatives and an aminosugar
EP1496917B1 (fr) * 2002-04-19 2005-12-07 Astion Development A/S Combinaison d'agonistes de recepteur adrenergique beta-2 et d'un sucre amine et leur utilisation pour le traitement de troubles immunomodulateurs
EP1871392A4 (fr) * 2005-04-01 2010-07-28 Christopher Richard Parish Régulation de l'angiogenèse par le biais de facteurs nod tels que les oligosaccharides de type glucosamine
EP1878789A4 (fr) * 2005-04-14 2008-08-27 Japan As Represented By Genera Cellule souche dérivée de graisse et son utilisation
ES2298052B1 (es) * 2006-08-03 2009-09-28 Bioiberica S.A. Procedimiento para la proliferacion de celulas.
ES2325392B1 (es) * 2007-12-28 2010-06-24 Bioiberica, S.A. Composicion para el tratamiento de la artrosis.
WO2012097052A2 (fr) * 2011-01-11 2012-07-19 Intertech Bio, Llc Composés à base de monosaccharides pour le traitement de maladies dermatologiques prolifératives et inflammatoires
WO2016137963A1 (fr) * 2015-02-25 2016-09-01 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Thérapies d'augmentation de sialylation pour maladies associées au stress oxydatif
CN109108275B (zh) * 2017-06-22 2022-05-03 国家纳米科学中心 氨基糖修饰的抗菌金纳米颗粒、其制备方法及应用

Also Published As

Publication number Publication date
IL302158A (en) 2023-06-01
EP4232454A4 (fr) 2024-09-18
AU2021365844A1 (en) 2023-06-08
KR20230091119A (ko) 2023-06-22
US20230390315A1 (en) 2023-12-07
AU2021365844A9 (en) 2025-03-13
JP2023545826A (ja) 2023-10-31
WO2022087107A1 (fr) 2022-04-28
CA3195941A1 (fr) 2022-04-28
MX2023004525A (es) 2023-05-25
CN116507340A (zh) 2023-07-28

Similar Documents

Publication Publication Date Title
Zhong et al. Inhibition of protein glycosylation is a novel pro-angiogenic strategy that acts via activation of stress pathways
CA2946825C (fr) Inhibiteur de l'atrophie musculaire contenant un glycoside de quercetine
Zhang et al. Parthenolide, an NF-κB inhibitor, alleviates peritoneal fibrosis by suppressing the TGF-β/Smad pathway
AU2015242791B2 (en) Drug Delivery Enhancer Comprising Substance For Activating Lysophospholipid Receptors
EA023244B1 (ru) Способ предотвращения старения клеток
MXPA06006651A (es) Metodos para suprimir una respuesta inmune o un tratamiento para un trastorno proliferativo.
KR102174166B1 (ko) 호모해링토닌을 유효성분으로 함유하는 세포노화 관련 질환 예방 또는 치료용 조성물
US20230390315A1 (en) Compositions and methods for treating ischemic conditions
US20220339233A1 (en) Compositions and methods for preventing recurrence of cancer
US20250275993A1 (en) Glycosylation inhibitors as therapeutics for stroke
WO2015093901A1 (fr) Composition pharmaceutique contenant un composant d'induction d'activation de la protéine taz pour différenciation musculaire et régénération musculaire
Cuijpers et al. Nutritional strategies targeting age-related skeletal muscle fibrosis: underlying mechanisms
Choudhury et al. Inhibition of protein glycosylation is a novel pro-angiogenic strategy that acts via activation of stress pathways
EP2446886A1 (fr) Régulateurs d'autophagie et leurs utilisations dans le traitement de dystrophies musculaires dues à un défaut du collagène VI
DK2964242T3 (en) Compositions for the treatment of cancer using gliadin peptides
EP3634582A1 (fr) Procédés et compositions pour le traitement de troubles de l'hyperpigmentation
EP4578459A1 (fr) Produit pharmaceutique pour le traitement et/ou la prévention du cancer
EP4578458A1 (fr) Médicament pour traitement et/ou prévention du cancer
KR101703637B1 (ko) Egfr 및 강글리오시드 n-글리콜릴 gm3 (neugcgm3)를 발현하는 종양의 치료를 위한 약학 조성물
KR20240054851A (ko) 티플락스티닌을 포함하는 복막 섬유증의 개선, 예방 또는 치료용 조성물
WO2018215628A1 (fr) Composés de type sulfonylurée dans le traitement d'une maladie associée à un dommage induit par uv
KR20240042415A (ko) 암의 치료 및/또는 예방을 위한 의약품
KR101134457B1 (ko) 호중구의 미생물 사멸활성을 강화하는 글라이신
JP2022112968A (ja) アミロイドβの凝集抑制剤、アミロイドβ凝集疾患用医薬組成物、およびその用途
HK40032147B (en) Activity modulator

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20230519

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 40099601

Country of ref document: HK

REG Reference to a national code

Ref country code: DE

Ref legal event code: R079

Free format text: PREVIOUS MAIN CLASS: C07H0013040000

Ipc: A61K0031702400

A4 Supplementary search report drawn up and despatched

Effective date: 20240821

RIC1 Information provided on ipc code assigned before grant

Ipc: C07H 15/04 20060101ALI20240815BHEP

Ipc: C07H 13/04 20060101ALI20240815BHEP

Ipc: A61K 45/06 20060101ALI20240815BHEP

Ipc: A61P 9/00 20060101ALI20240815BHEP

Ipc: A61K 31/7024 20060101AFI20240815BHEP